A methanol assimilating yeast, i.e. methylotrophic yeast, is able to grow on methanol as a sole carbon source. In the initial reaction of methanol metabolism by the methylotrophic yeast, formaldehyde and hydrogen peroxide are produced from methanol and oxygen by alcohol oxidase. The produced hydrogen peroxide is catabolized into water and oxygen by catalase. The formaldehyde is oxidized to carbon dioxide through the actions of formaldehyde dehydrogenase, S-formylglutathione hydrolase, and formate dehydrogenase. NADH formed upon these oxidations becomes an energy source of the yeast cell. Simultaneously, due to the action of dihydroxyacetone synthase, the formaldehyde condenses with xylulose-5-phosphate, and the condensate product is converted into glyceraldehyde-3-phosphate and dihydroxyacetone, which become components of the yeast cell via pentose phosphate cycle. When the methylotrophic yeast is cultured in the presence of methanol, the above-mentioned alcohol oxidase, dihydroxyacetone synthase and formate dehydrogenase are produced in an extremely large amount, reaching about 40% of the intracellular soluble proteins.
As described above, the methylotrophic yeast is considered to be a suitable host for expression system of heterologous genes in that it can be mass-cultured with inexpensive methanol and that it has a promoter for methanol-metabolizing enzyme with a strong transcriptional activity unfound in other yeasts.
Candida boidinii is a kind of methylotrophic yeast. Utilizing this yeast, methods for expressing a heterologous gene by using regulatory regions of alcohol oxidase gene and formate dehydrogenase gene have been studied (JP-A-5-344895, WO97/10345, etc.). In most cases of such an expression system, a heterologous gene is produced in a larger amount within a transformant having the chromosome into which a higher copy number of an expression vector has been integrated (Appl. Microbiol. Biotechnol., 42, 860-864 (1995); Proceedings of Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry, 1997, p. 257; and Proceedings of Annual Meeting of Society for Bioscience and Bioengineering, Japan, 1997, p. 314). Considering the stability of an expression vector within a transformant, it seems to be more desirable to achieve a high amount of expression at a low copy number, and there has been a demand for developing promoters with stronger transcriptional activity.